Accepted article

Rev. sci. tech. Off. int. Epiz., 2014, 33 (3), ... - ...
The equine arteritis virus isolate from the
2010 Argentinian outbreak
This paper (No. 22102014-00048-EN) has been peer-reviewed, accepted, edited, and
corrected by authors. It has not yet been formatted for printing. It will be published in
December 2014 in issue 33-3 of the Scientific and Technical Review
G.E. Metz (1, 2), M.S. Serena
M.G. Echeverría (1, 2)*
(3)
, C.J. Panei
(2, 3)
, E.O. Nosetto
(1)
&
(1) Virology, Faculty of Veterinary Sciences, National University of
La Plata, 60 and 118, CC 296, 1900 La Plata, Argentina
(2) Members of CONICET-CCT (Consejo Nacional de
Investigaciones Científicas y Técnicas – Centro Científico
Tecnológico), La Plata, Argentina (Scientific Research Council)
(3) Immunology, Faculty of Veterinary Sciences, National University
of La Plata, 60 and 118, CC 296, 1900 La Plata, Argentina
*
Corresponding author: [email protected];
[email protected]
Summary
A semen sample from a stallion infected during the 2010 equine
arteritis virus (EAV) outbreak was received for viral isolation prior to
castration of the animal. The virus was identified using a polyclonal
antibody immunofluorescence test. Reverse-transcription polymerase
chain reaction (RT-PCR) was used to amplify a region of the GP5
gene with primers GL105F and GL673R. The PCR products were
purified and sequences of both strands were determined in a
MegaBACETM 1000 with inner primers CR2 and EAV32. A
phylogenetic dataset was built with the previously reported sequences
of five strains isolated in Argentina, together with a group of selected
sequences obtained from GenBank. The unrooted neighbour-joining
tree was constructed using molecular evolutionary genetic analysis
(MEGA) and bootstrap analyses were conducted using 1,000 replicate
datasets. Evolutionary distances were computed using the maximum
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composite likelihood method. A NetNGlyc server analysis at the
Technical
University
of
Denmark
(www.cbs.dtu.dk/services/NetNGlyc/) was used to predict Nglycosylation in GP5 sequences. The phylogenetic analysis revealed
that the new strain (GLD-LP-ARG), together with other strains
previously isolated, belongs to the European group EU-1 but in a
different branch. The new strain shows 99% nucleotide identity with
strain A1 and 98.1% with the Belgian strain 08P178. Persistently
infected stallions and their cryopreserved semen constitute a reservoir
of EAV, which ensures its persistence in the horse population around
the world. These findings reinforce the importance of careful
monitoring of persistently infected stallions, as well as semen straws,
by RT-PCR or test mating, in accordance with national regulations.
Keywords
Argentina – Equine arteritis virus – Outbreak – Semen sample.
Introduction
Equine arteritis virus (EAV) was first isolated from fetal lung tissue
during an outbreak of respiratory disease and abortion in Bucyrus,
Ohio, in the United Stated of America (USA) (1). Until 2010, the
prevalence of EAV-infected stallions resident in Argentina was
thought to be very low. The first report of serological evidence was in
1984 (2), when a prevalence of 9.2% was found in the population of
warmblood horses sampled. The virus (strain LP01) was first isolated
in 2001 (3); later, strain LT-LP-ARG was isolated from the testicle of
a seropositive stallion that had been imported into Argentina in 1998
(4). In 1998, several EAV antibody-positive animals were detected in
two sport-horse breeding farms that practised artificial insemination
with imported semen. A follow-up study between July 2001 and
December 2003 found a prevalence of 45.8% in one of those farms;
three stallions were virus isolation-positive (strains LP02/R, LP02/C,
LP02/P) (5). Three new sequences of EAV from three archive semen
samples were obtained in 2008: one (RO-LP-ARG) from a stallion
housed on the breeding farm where the first strain of EAV was
isolated and the other two (RZ-LP-ARG, KB-LP-ARG) from the farm
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where the LP02 strains had been isolated (6). Before the 2010
occurrence of equine viral arteritis in Argentina, the virus had not
been involved in respiratory disease, abortion or foal death; the
prevalence was restricted to sport-horse breeds and to certain breed
lineages (7). Phylogenetic investigations of the virus have focused on
the hypervariable region of the GP5 gene, in both European and North
American strains (8, 9, 10). At present, Argentinian EAV strains all
belong to the same group (6, 11, 12). Although infrequently reported
in the past, confirmed outbreaks of arteritis appear to be on the
increase and in the past decade the disease has been reported in the
USA (13), France (14) and Belgium (15). Analysis of phylogenetic
relationships has been demonstrated to be an effective tool in tracing
the source of EAV infection.
In 2010, two mares on a thoroughbred breeding farm in Buenos Aires
Province seroconverted after insemination with frozen semen
imported from the Netherlands. The semen straws from the stallion
believed to be responsible for spreading the infection were submitted
to the Virology Laboratory, National Institute of Agricultural
Technology, Castelar, Buenos Aires, and EAV was isolated (16). Four
other farms that had used this semen were put into quarantine and all
the mares that had been inseminated with the infective semen were
found to be seropositive.
A high prevalence of EAV infection was also found at an equestrian
club located in central Buenos Aires, where two other mares had been
inseminated with infective semen from a stallion housed at the club.
Respiratory disease, fever, limb oedema and abortions were observed
during this outbreak. Twenty-seven jumping stallions seroconverted
and 22 persistently infected stallions were castrated. In the present
study, the ORF5 region of this EAV strain was analysed genetically
and compared with sequences of other strains from Argentina and
elsewhere.
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Materials and methods
Sample processing and virological methodology
The infected stallion at the equestrian club in central Buenos Aires
was a Hannoverian born in 2000 that had been housed at the club
since 2006; the horse was found to be seropositive in May 2010.
Before castration of the animal, a semen sample was processed for
routine virus isolation at the Virology Laboratory, Faculty of
Veterinary Sciences, National University of La Plata, as described
previously (3, 17). Briefly, serial tenfold dilutions (10–1 to 10–3) of the
sample were inoculated in duplicate onto RK13 cells grown in 6-well
plates. The cells were maintained in culture medium containing 2%
fetal bovine serum. Plates were incubated at 37°C in an atmosphere of
5% CO2 and examined daily for cytopathic effects. The virus was
identified using a polyclonal antibody immunofluorescence test, as
described previously (3).
Reverse-transcription polymerase chain reaction and
sequencing
Viral RNA was extracted from 500 µl of the supernatant of infected
RK13 cells with 500 µl of TRIzol® (Invitrogen) and precipitated with
isopropanol. Portions (5 µl) of RNA resuspended in distilled water
were used for cDNA synthesis using reverse transcriptase and random
hexamers (Promega). For PCR amplification, the primers GL105F and
GL673R, which flank a 546-nt region of the GP5 gene, were used
(18). Denaturation, annealing and extension consisted of 35 cycles at
94°C for 45 s, 60°C for 1 min and 72°C for 90 s, respectively. The
PCR products were run on 2% agarose gels, stained with ethidium
bromide, observed under UV light and purified using a PCR
purification kit. The sequences of both strands were determined in a
MegaBACETM 1000 with primers CR2 and EAV32, which flank a
519-nt region (8).
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Dataset and phylogenetic analysis
The phylogenetic dataset was built with five strains isolated in
Argentina, sequences reported previously (3, 4, 5, 6, 11), and a group
of selected sequences obtained from GenBank (Table I). Sequences
were edited using BioEdit software version 5 (19) and aligned in
molecular evolutionary genetic analysis (MEGA) software, version
4.0, using the ClustalW algorithm. Sequence pair distances were
calculated by DNAstar® (20). The unrooted neighbour-joining tree
was constructed using MEGA and bootstrap analyses were conducted
using 1,000 replicate datasets. Evolutionary distances were computed
using the maximum composite likelihood method (20).
Analysis of N-glycosylation sites
A NetNGlyc server analysis at the Technical University of Denmark
(www.cbs.dtu.dk/services/NetNGlyc/) was used to predict Nglycosylation in GP5 sequences. This program predicts asparagines to
be N-glycosylated according to the Asn-Xaa-Ser/Thr sequons (where
Xaa is not Pro), with a threshold of 0.5.
Results
Viral isolation and reverse-transcription polymerase chain
reaction
After two passages in confluent monolayers of RK13 cells, cytopathic
effects were observed in cells inoculated with the seminal plasma at
10–1 and 10–2 dilutions when compared with control cells. The isolated
virus strain was named GLD-LP-ARG. After reverse transcription,
cDNA was obtained from the supernatants of the cultures. An
immunofluorescence test confirmed the presence of EAV antigen; no
virus-specific fluorescence was observed in control mock-infected
cells. Using primers specific for the GP5 gene, the cDNA gave a
visible 591-bp band in an ethidium bromide-stained agarose gel. No
bands were observed in the negative control used in the PCR. The
519-bp sequence was analysed and aligned with the Argentinian EAV
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isolates and sequences previously reported, and also with American
and European reference strains.
Phylogenetic analysis
The partial nucleotide sequence data for gene GP5 reported here have
been previously submitted to GenBank under accession number
JQ316510. The distance tree obtained by the neighbour-joining
algorithm showed that all the Argentinian sequences, except for the
first isolate (LP01), are clustered together with high bootstrap values.
However, the new isolate, GLD-LP-ARG, is closely related to strains
P1, R1, A1, G1, H20, H22, F23 and 08P178, with LP01 as the most
basal strain of EAV in the EU-1 subgroup (Fig. 1). Use of other
algorithms such as maximum likelihood revealed the same topology:
one group comprises the LP02 strains (LP02/R, LP02/C, LP02/P), LTLP-ARG, RZ-LP-ARG, RO-LP-ARG and KB-LP-ARG; another
comprises some European strains and GLD-LP-ARG, with the LP01
strain being the most basal of the group (Fig. 1). The percentage
identity among the EAV sequences from the EU-1 clade where the
Argentinian strains are located varied between 87.8% and 100%.
Strain GLD-LP-ARG shares 99% nucleotide identity with strain A1
and 98.1% with the Belgian strain 08P178.
Analysis of N-glycosylation sites
Changes in GP5 in the Argentinian sequences are described below.
The deduced amino acid sequences (51–222) of the variable and
conservative regions of the protein are illustrated in Fig. 2. There are
no deletions or insertions. In the first constant region (C1), the only
substitution that was found had already been reported (position 57 Cys
x Trp in strain LP02/R) (12). The second constant region (C2)
remained invariable in all the sequences and the third constant region
(C3) showed only substitution in KB-LP-ARG (position 199 Ala x
Thr). Analysis of the V1 region (amino acids 61–121) showed that
Cys in positions 63, 66 and 80 remained invariable in all the
Argentinian sequences. As previously reported (12), neutralising site
C (amino acids 67–90) was hypervariable. However, one change
(position 70 Asp x Ser) was new for sequences in the 2010 outbreak
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strain (GLD-LP-ARG). In contrast, Asp x Glu (position 71) appeared
in both GLD-LP-ARG and LP01 (the first strain isolated in
Argentina), and Glu x Gly (position 93) appeared in KB-LP-ARG.
Regarding amino acids 81–84, when analysing the putative N-linked
glycosylation sites, all the sequences showed asparagine sites at amino
acid 81 (variable) and in position 56 (conserved and critical for virus
infectivity). It has been reported that loss of glycosylation sites in
other arteriviruses can alter the virulence and tropism of the virus but
it is not known whether these changes play an analogous role in EAV
(21). However, according to the prediction glycosylation program
used here, the asparagines present in position 81 were not sufficient to
glycosylate in strains LP01 and LP02/P.
Discussion
Most EAV strains belong to one of three large genetic groups (EAV-1,
EAV-2, EAV-3) (18). This classification has been modified recently
as a North American clade (NA, formerly EAV-2) and a European
clade (EU) with two subgroups (EU-1 and EU-2, formerly EAV-1 and
EAV-3 respectively) (21). This approach has also been used to
construct the phylogeny of isolates in Poland (22), where 44 isolates
are included in the European subgroups (EAV-1 or EAV-3). The new
Argentinian strain shares similarity with several European
phylogenetic strains such as H20 and H22 (Hungary), F23 (France)
and 08P178, the last strain isolated from an outbreak in Belgium, as
well as with P1, R1, A1 and G1 isolated in the USA (15). Strain S-113
from the Netherlands belongs to the North American group. To date,
none of the Argentinian strains has been linked to the North American
EAV strains. Differences are mostly localised in the V1 region, in
particular within the neutralisation sites B, C and D. As previously
reported, South African donkey isolates were classified in subgroup
EU-2 but formed only one cluster (21). Furthermore, six isolates from
Lipizzaner stallions in South Africa belonged to subgroup EU-1 and
did not cluster with the South African asinine strains, thus
representing a unique variant (21). As expected, some sequences, such
as H21, SWZ64, S3 and S4, did not form statistically supported
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clusters and therefore formed an unclassifiable group of highly
variable sequences branching off near the centre.
According to data from the Argentinian authorities, EAV was isolated
from the imported semen of a stallion in the Netherlands. Some
Argentinian premises were infected as a consequence of using the
infective semen or through the movement of infected horses without
observation of clinical signs after spread of the virus via the
respiratory route. In the present study, a stallion housed in an
equestrian club was castrated because it was persistently infected and
shedding the virus in semen. The prevalence of infection at the club
was very high (80%). The genetic characterisation reported here
confirms that the strain from the 2010 Argentinian outbreak belongs to
subgroup EU-1, together with all the Argentinian sequences reported
to date.
Previously, all Argentinian isolates were related to a non-pathogenic
strain and this is the first report of an EAV isolate associated with
clinical signs in Argentina. Regarding virulence, EAV strains can be
classified as velogenic, mesogenic or lentogenic, according to the
clinical signs (23): velogenic strains cause fatal disease in adult
horses, mesogenic strains cause less severe disease and lentogenic
strains do not cause any clinical signs. Loss of virulence in EAV has
been related to the amino acids sequences of structural and
nonstructural proteins, and eight critical amino acid substitutions in
the structural protein GP5 have been identified as being responsible
for EAV attenuation (23). The same amino acid substitutions were
observed in the present study when comparing velogenic and
mesogenic strains (23). Although the amino acid substitutions of the
2010 EAV Argentinian isolate related to a high- or medium-virulence
strain, analysis of strain virulence is more complex; GP5 analysis
alone provides an approximation, but analysis of other structural and
nonstructural proteins is required. Further, host genetic factors such as
the haplotype of the equine could determine resistance or
susceptibility to EAV infection and thus influence the capability of the
virus to generate clinical signs (24).
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In summary, the EAV-infected equine population in Argentina in
2010 showed mild clinical signs that could be correlated by analysing
the sequence of gene GP5. Using amino acid substitution, the 2010
isolate was grouped with velogenic or mesogenic strains (23).
According to the phylogenetic study, this strain has high similarity
with the Belgian strain 08P178, also reported as a European subtype
of low virulence (25). The Belgian strain was used recently in an
experimental infection in naïve ponies (26) where it produced mild
clinical signs when compared with North American strains (27). It
was concluded that the differences in clinical signs may be dependent
on both the viral strain and the breed of animal (26).
Stallions and their cryopreserved semen constitute a reservoir of EAV,
thus ensuring persistence of the virus in the horse population around
the world and causing new outbreaks such as that in Argentina in
2010. These findings reinforce the importance of careful monitoring
of EAV in persistently infected stallions, as well as in semen straws,
by RT-PCR or test mating, according to national regulations.
Although most EAV strains are of low virulence, outbreaks with
severe clinical signs may occur.
Conclusion
The new Argentinian strain shows 99% nucleotide identity with strain
A1 and 98.1% with the Belgian strain 08P178.
Acknowledgements
The assistance of Ms A. Conde and Mr C. Leguizamon is very
gratefully acknowledged.
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Table I
Origins, names and phylogenetic groups of the equine arteritis
virus strains used in this study
Country of
isolation
GenBank accession
no.
Phylogenetic
group
F5
France
EF492543
NA
F6
France
EF492544
NA
F7
France
EF492545
NA
F8
France
EF492546
NA
F9
France
EF492547
EU-1
F10
France
EF492548
EU-2
F11
France
EF492549
NA
F12
France
EF492550
NA
F13
France
EF492551
NA
F14
France
EF492552
NA
F15
France
EF492553
EU-2
F23
France
EF492561
EU-1
F24
France
EF492562
NA
F25
France
EF492563
NA
F26
France
EF492564
NA
H20
Hungary
AY453305
EU-1
H21
Hungary
AY453306
EU-1
H22
Hungary
AY453307
EU-1
H23
Hungary
AY453308
EU-1
H24
Hungary
AY453309
EU-2
I13
Italy
AY453310
EU-1
I14
Italy
AY453311
NA
I15
Italy
AY453312
NA
I16
Italy
AY453313
EU-1
I17
Italy
AY453314
EU-1
I18
Italy
AY453315
EU-1
I19
Italy
AY453316
NA
I20
Italy
AY453317
EU-1
PLA00-1
Poland
EF102348
EU-1
PLB01-1
Poland
EF102349
EU-1
PLG02-1
Poland
EF102354
EU-1
PLK02-8
Poland
EF102363
EU-1
Virus strain
PLH05-1
Poland
EF102355
EU-1
J25-941109-3
South Africa
AY956603
EU-2
J7-931125
South Africa
AY956602
EU-2
J6-940309
South Africa
AY956601
EU-2
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J5-940309
South Africa
AY956600
EU-2
J4-931209
South Africa
AY956599
EU-2
J3-931209
South Africa
AY956598
EU-2
J2-931125
South Africa
AY956597
EU-2
J1-931125
South Africa
AY956596
EU-2
RSA1
South Africa
AY453332
EU-2
RSA2
South Africa
AY453333
NA
RSA3
South Africa
AY453334
EU-1
RSA4
South Africa
AY453335
EU-1
RSA5
South Africa
AY453336
EU-1
RSA6
South Africa
AY453337
EU-1
RSA7
South Africa
AY453338
EU-1
RSA8
South Africa
AY453339
EU-1
S2
Sweden
AY453340
EU-2
S3
Sweden
AY453341
EU
S4
Sweden
AY453342
EU
S5
Sweden
AY453343
EU-2
S6
Sweden
AY453344
EU-2
S7
Sweden
AY453345
EU-2
S-113
Holland
AF099833
NA
S-544
New Zealand
AF099834
NA
SWZ64
Switzerland
U38609
EU
KY84
USA
AF107279
NA
KY93
USA
U81017
NA
VBS53
USA
U81013
NA
D84
USA
AF107266
NA
E85
USA
AF107275
NA
G1
USA
AF118777
EU-1
P1
USA
AF118775
EU-1
R1
USA
AF118773
EU-1
A1
USA
AF118769
EU-1
MT89
USA
U38604
NA
08P178
Belgium
JN254761
EU-1
LP01
Argentina
DQ435439
EU-1
LP02/R
Argentina
DQ435440
EU-1
LP02/C
Argentina
DQ435441
EU-1
LP02/P
Argentina
DQ435442
EU-1
LT-LP-ARG
Argentina
EU622859
EU-1
KB-LP-ARG
Argentina
EU622860
EU-1
RZ-LP-ARG
Argentina
EU622861
EU-1
RO-LP-ARG
Argentina
EU622862
EU-1
GLD-LP-ARG
Argentina
JQ316510
EU-1
No. 22102014-00048-EN
15/18
Rev. sci. tech. Off. int. Epiz., 33 (3)
16
Fig. 1
Maximum likelihood phylogenetic tree based on analysis of ORF5
nucleotide sequences in equine arteritis virus strains
Bootstrapping of the tree was carried out in 1,000 duplicates using the
MEGA 4.0 software program
EU-1
No. 22102014-00048-EN
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Rev. sci. tech. Off. int. Epiz., 33 (3)
No. 22102014-00048-EN
17
17/18
Rev. sci. tech. Off. int. Epiz., 33 (3)
18
Fig. 2
Alignment of deduced amino acids for partial GP5 sequences in
Argentinian equine arteritis virus strains
Non-consensus amino acids are in bold letters. Variable and conserved
regions are indicated with capital letters. Neutralisation sites B, C and
D are indicated in boxes. Predicted N-glycosylation sites are
underlined
Neut. Site B
Neut. Site C
____
_________________________
C1
RO-LP-ARG
KB-LP-ARG
LT-LP-ARG
LP02/R
RZ-LP-ARG
LP02/C
LP02/P
GLD-LP-ARG
LP01
51
HTALYNCSAS
HTALYNCSAS
HTALYNCSAS
HTALYNWSAS
HTALYNCSAS
HTALYNCSAS
HTALYNCSAS
HTALYNCSAS
HTALYNCSAS
****** ***
V1
C2
KTCWYCEFLDDQIITFGTGCNDTYSVPVSTVLEQAHGPYSVLFDDMPPFIYYGREFGIFVM
KTCWYCEFLDDQIITFGTGCNDTYSVPVSTVLGQAHGPYSVLFDDMPPFIYYGREFGIFVM
KTCWYCEFLDDQIITFGTGCNDTYSVPVSTVLEQAHGPYSVLFDDMPPFIYYGREFGIFVM
KTCWYCEFLDDQIITFGTGCNDTYSVPVSTVLEQAHGPYSVLFDDMPPFIYYGREFGIFVM
KTCWYCEFLDDQIITFGTGCNGTYSVPVSTVLEQAHGPYSVLFDDMPPFIYYGREFGIFVM
KTCWYCVFLDDQIITFGTGCNDTHSVPVSTVLEQAHGPYSVLFDDMPPFIYYGREFGIFVM
KTCWYCEFLDDQIITFGTGCNNTHSVPVSTVLEQAHGPYSVLFDDMPPFIYYGREFGIFVM
KTCWYCEFLSEQIITFGTGCNDTYSVPVSTVLEQAHGPYSVLFDDMPPFIYYGREFGIFVM
ETCWYCVFLDEQVITFGTGCNNTYSVPVSTVLEQAHGPYSVLFDDMPPFIYYGREFGIFVM
:***** **.:*:********.*:******** ****************************
V2
RO-LP-ARG
KB-LP-ARG
LT-LP-ARG
LP02/R
RZ-LP-ARG
LP02/C
LP02/P
GLD-LP-ARG
LP01
Neut. Site D
__________
ATLILEMCVSILFVVYGLYSGAYLAMGIFATTLVVHSV
ATLILEMCVSILFVVYGLYSGAYLAMGIFATTLVVHSV
ATLILEMCVSILFVVYGLYSGAYLAMGIFATTLVVHSV
ATLILEMCVSILFVVYGLYSGAYLAMGIFATTLVVHSV
ATLILEMCVSILFVVYGLYSGAYLAMGIFATTLVVHSV
ATLILEMCVSILFVVYGLYSGAYLAMGIFATTLVVHSV
ATLILEMCVSILFVVYGLYSGAYLAMGIFATTLVVHSV
ATLILEMCVSILFVVYGLYSGAYLAMGIFATTLVVHSV
VTLILEMCVSILFVVYGLYSGAYLAMGIFATTLVVHSV
.*************************************
No. 22102014-00048-EN
C3
DVFMFYPVLVLFFLSVLPY
DVFMFYPVLVLFFLSVLPY
DVFMFYPVLVLFFLSVLPY
DVFMFYPVLVLFFLSVLPY
DVFMFYPVLVLFFLSVLPY
DVFMFYPVLVLFFLSVLPY
DVFMFYPVLVLFFLSVLPY
DVFMFYPVLVLFFLSVLPY
DVFMFYPVLVLFFLSVLPY
*******************
V3
VVLRQLLWLCLAWRYRCTLHASF
VVLRQLLWLCLAWRYRCTLHTSF
VVLRQLLWLCLAWRYRCTLHASF
VVLRQLLWLCLAWRYRCTLHASF
VVLRQLLWLCLAWRYRCTLHASF
VVLRQLLWLCLAWRYRCTLHASF
VVLRQLLWLCLAWRYRCTLHASF
VVLRQLLWLCLAWRYRCTLHASF
VVLRQLLWLCLAWRYRCTLHASF
********************:**
ISAEGKIYPVDPGLPIAAAGN
ISAERKIYPVDPGLPIAAAGN
ISAEGKIYPVDPGLPIAAAGN
ISAEGKIYPVDPGLPIAAAGN
ISAEGKIYPVDPGLPIAAAGN
ISAEGKIYPVDPGLPIAAAGN
ISAEGKIYPVDPGLPIAAAGN
ISAEGKIYPVDPGLPIAAAGN
ISAEGKIYPVDPGLPIAAAGN
**** ****************
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